TC1 deletion PCR: artifacts

[Ron Kagan]

In article <01HFZEV47GBM8WWBDY at acs.wooster.edu> William R. Morgan,
wmorgan at ACS.WOOSTER.EDU writes:
>I'm conducting a screen for Tc1-mediated deletions starting with a Tc1
>insertion allele of kin-16 and I'm getting a major artifactual product of
>about 1 kb.  Thomas Burglin and David Greenstein state that they see
>similar "non-specific bands that are also present in all reactions, but
you
>should look for bands that occur only in one of the samples" (Burglin and
>Greenstein, "Tc1 Deletion PCR Tips" available by Gopher at
>weeds.mgh.harvard.edu).  However, I'm concerned that the amount of the
>artifactual product (typically 25-75 ng/ 10 ul) may be out-competing the
>desired, rare deletion products.  Should I worry about this PCR artifact
or
>should I merrily continue my screen?

I don't know if this is feasible with the number of samples you may be
working with, but could you prepare restriction digest quality DNA? 
Then, prepare some of the 1 Kb artifact DNA, find a restriction enzyme
that cuts the 1 Kb artifact but is not likely to cut your desired
product.  You could then treat your samples with that restriction enzyme
prior to doing PCR, thus eliminating the 1 Kb artifact.



Ron Kagan

**********************************************************************
"My Kindness is not random, and nothing senseless was ever beautiful."

                                                 - J. Zabriskie
**********************************************************************
Ron Kagan
rkagan at ewald.mbi.ucla.edu